Riboswitches are natural cis-acting RNA genetic regulatory elements. They bind metabolites and have critical roles in the regulation of basic metabolic pathways including purine, amino acid and vitamin biosynthesis. Riboswitches use a variety of different mechanisms to regulate gene expression upon ligand binding including modulation of transcription termination, translation suppression, and self-cleavage. From the few studies of riboswitch mechanism of action, the kinetics of ligand binding and RNA transcription play an important role in the regulation of gene expression. The specific aims of this proposal are to 1) characterize the cooperative glycine riboswitch mechanism of action and 2) construct kinetic models of riboswitch RNA folding. Through a series of biophysical measurements, the ligand binding kinetics of the glycine riboswitch will be measured and how the rate of transcription may affect the behavior of the switch will be explored. Prospective transcriptional pause sites within the switch will be examined to further study how ligand binding affinity may differ for discreet intermediates compared with the entire switch. Additionally, the sequence determinants for the cooperative behavior will be explored by targeting the phylogenetically conserved linker between the two glycine binding motifs. Secondary structure models exist for all the riboswitches based on phylogenetic and thermodynamic analysis. However, most riboswitches examined have slow ligand binding kinetics indicating an induced fit mechanism. The kinetic accessibility of secondary structure conformations necessary for ligand binding will be examined using existing RNA secondary structure folding simulations. It is anticipated that some transcriptional intermediates allow faster folding than others. It is anticipated that this modeling may lead to the development of better computational design strategies for engineered RNA gene regulatory elements. Due to their widespread appearance in prokaryotes and regulation of essential metabolic processes, riboswitches represent new targets for antimicrobial agents. Additionally, riboswitches are natural (and often better) examples of RNA elements engineered for synthetic biology applications. Understanding riboswitch mechanism of action also will lead to better design of engineered RNA elements.